Method for obtaining polyfructosans

ABSTRACT

A kidney function diagnostic agent comprising high water-soluble polyfructosans derived from the storage organs of monocotyledon plants.

United States Patent Nitsch et a1.

July 8, 1975 METHOD FOR OBTAINING POLYFRUCTOSANS Inventors: ErnstNitsch; Siegried Muhlbock, both of Linz, Austria Assignee:Laevosan-Gesellschaft mlbH & Co.

KG, Linz, Austria Filed: Dec. 6, 1971 App]. No.: 205,293

Foreign Application Priority Data References Cited UNITED STATES PATENTS5/1958 I-Iill 260/209 R OTHER PUBLICATIONS Schlubach et al., Chem. Abst,Vol. 51, 1957, pp. 16310g16311b.

Schlubach et al., Chem. Abst., Vol. 52, 1958, p. 3698 d.

Schlubach et al., Chem. Abst, Vol. 52, 1958, pp. 19,963 i l9,965 0.

Primary Examiner-Johnnie R. Brown Attorney, Agent, or Firm-Sughrue,Rothwell, Mion, Zinn & Macpeak [57] ABSTRACT A kidney functiondiagnostic agent comprising high water-soluble polyfructosans derivedfrom the storage organs of monocotyledon plants.

7 Claims, N0 Drawings METHOD FOR OBTAINING POLYFRUCTOSANS BACKGROUND OFTHE INVENTION 1. Field of the Invention This invention relates to a newkidney diagnostic agent which, when administered parenterally, isneither degradated nor altered in the body and which is able to beeliminated through the kidneys. On the basis of the elimination speedand the quantity eliminated, one can draw conclusions as to the functionof the kidney.

2. Description of the Prior Art About 35 years ago, inulin, apolyfructosan from the storage organs of certain types of composites(for example, Inula helenium, Helianthus tuberosus, Cichorium intybus),were introduced into kidney diagnosis as a clearance substance for thedetermination of the size of the glomerulum filtrate; see Richards A.N., Westfall B. B. and Pott A. A. Proc. Exper. Biol. (N.Y.), 32, 73(1934); Shannon .1. A. and Smith H. W., Clin. Invest. 20, 169 (1935).For this purpose, a known volume of inulin solution is administeredparenterally and the elimination of the inulin is followed up with aperiodic concentration determination in the blood and the urine. Theprerequisite for this use is the property of inulin to be neitherstored, nor chemically altered in the body. In spite of certainpractical disadvantages, inulin has so far proved to be excellent forthis task as a standard substance. The chief disadvantage in the use ofinulin resides in the fact that it is difficult to dissolve in coldwater. The normally employed percent solution crystallizes at coldtemperatures, and is accompanied by the formation of a crustyprecipitate. Before it can be given to a patient, this precipitate mustonce again be dissolved through heat. At this point there is a dangerthat the inulin could be hydrolyzed into low molecular fractions andfructose, which in contrast to inulin, are broken down by the body; onthe other hand, after injection of insufficiently dissolved preparationsblockage of the thinner blood vessels of the body may occur which couldprecipitate serious physiological complications.

SUMMARY OF THE INVENTION In accordance with this invention, there is nowproposed, new kidney diagnostic agents, themselves longknown, comprisingthe watersoluble polyfructosans from the storage organs of certainmonocotyledon plants, and especially, from the families of the Liliaceaeand the Gramineae.

Thus, the object of this invention is to provide a kidney functiondiagnostic agent with a polyfructosan content, which is characterized bythe fact that it contains water-soluble polyfructosans easily soluble inwater from the storage organs of monocotyledon plants, and especiallyfrom the families of the Lilicaeae and Gramineae.

DETAILED DESCRIPTION OF THE INVENTION For reasons of economy, one takesinto prime consideration the use of Sinistrin, a polyfructosan from thesea onion (Urginea maritima) see Schmiedeberg O., l-loppe Seylers Z.Physio]. Ehem (Journal of Physiological Chemistry), 3, 1 12 (1879) andSchluback H. H. and Florshein W., Ber. dtsch. (Reports of the GermanChemical Society) 62, (1929), and the polyfructosan from garlic (Alliumsativum), as well as Graminin and Triticin, see Klein 6., Handbuch derPflanzenanalyse (Handbook of Plant Analysis), Spring 1932, 1st part,pages 866873, and Paech K. and Tracey M. V., Moderne Methoden derPflanzenanalyse (Modern Methods of Plant Analysis) Springer 1955, 2ndVolume, pages 184-196.

All of these polyfructosans are characterized in that l they are easilywater-soluble, (2) they are easily hydrolyzed by acids, (3) theiraverage molecular weight is about 3,000, corresponding to about 18monosaccharide units, and (4) their fructose residues are primarilyB-1,2-bound, see Schlubach H. H., Sing. 0. K., Liebigs Ann. (LiebigsYearbook) 544, 1 11 (1940). Furthermore, these compounds prove to becompletely non-toxic when tested clinically and pharmacologically.

For the production of the above-mentioned watersoluble polyfructosans,the methods described in the literature to date leave much to be desired(Schmiedeberg 0., I-Ioppe Seylers Z. Physio]. Chemistry 3, 112 (1879)and Klein G. Handbuch der Pflanzenanalyse, Springer 1932, 1st part,pages 866-873.) Basically, they reside in the preparation of a wateryextract from which a part of the ballast substance is removed throughprecipitation with vinegar of lead. This is followed by subsequentremoval of the lead and the polyfructosans are then precipitated as limeor barite compounds by the addition of milk of lime or baryta water.After filtering by suction and washing, they are broken down intocarbonic or oxalic acid, after which the polyfructosan solution thuspurified is concentrated into a syrup and precipitated with alcohol.Since the precipitation very stubbornly produces monosaccharides andother impurities, numerous re-precipitations are normally required inorder to attain a sufficient degree of purity.

A further object of this invention is to provide a method for theproduction of polyfructosans for use as a kidney function diagnosticagent, which comprises extracting the particular drugs with low alcoholsor with acetone having a water content of -40 percent. The extract ispreferably brought to a pH of 9 with calcium hydroxide, filtered, andconcentrated under reduced pressure, into a thick syrup, whereupon thethickened extract, after addition of about half its weight in a lowalcohol (preferably methanol) is aciditied with a strong acid(preferably hydrochloric acid) to a pH of between 1 and 3. Thepolyfructosan is then precipitated with a 2 to 8 fold weight of a lowalcohol (preferably methanol) or with acetone, and the precipitate thusobtained is converted into a powdery crystalline form through repeatedkneading with a water-free precipitation agent. If desired, the solutionof the polyfructosan thus obtained can be completely desalted viaion-exchange, treated with active carbon (active charcoal), filtered,and then either filled, after removal of the organic solvent residues bymeans of distillation, directly into ampoules, or optionally it can bedried, after concentration into a syrup consistency, in a vacuum, to athin layer.

The method just described is best carried out as follows:

The coarsely comminuted plant material is repeatedly extracted withaqueous alcohols or acetone (preferably methanol, ethanol, orisopropanol) or with acetone having 80-40 percent water by means ofpercolation, if necessary, at increased temperatures. The function ofthe organic solvent is (1) to prevent degradation by microorganisms, (2)to suppress the effect of the polyfructosan-decomposing ferments, and(3) to prevent the dissolution of interfering pectin and shinysubstances. The extracts thus obtained are alkalized to a pH value of 9with milk of lime (calcium hydroxide), filtered and the resultantfiltrate is evaporated in a vacuum to produce a syrup of about 70-80percent dry substance content. After cooling, it is, portion by portion,mixed with so much of a low alcohol illustrated above (preferablymethanol) or with acetone until no remaining precipitate develops;concentrated hydrochloric acid is then added to obtain a pH of l to, 3'and preferably 2.5. Next, we add a sufficient amount of solvent (sameas above) to complete precipitation. 1n adding the acid, we assure thata large part of the existing salts and dyes, which otherwise would alsobe precipitated, remain in solution. The precipitate, which consists ofa white, viscous mass, is separated from the supernatant liquid and isthoroughly kneaded several times with small portions of solvent, therebyforming a fine-grained crystalline product which finally is liberatedfrom the adhering liquid by centrifuge or suction filter. The substancethus obtained is already very pure but, for parenteral application, itmust be dissolve again in water and further purified by'means of ionexchange and filtration of the desalted solution with active carbon.Finally, the solution thus obtained can either be filled directly intoampoules after removal of the solvent residues through distillation andadjustment' of the EXAMPLE 1 Productionof Polyfructosan (Sinistrin) Fromthe Onion Bulbs of the Composite Urginea Maritima 100 Kg of commerciallyavailable dried red sea onion were coarsely ground in a knife-mill witha screen insert having a 4-mm perforation diameter, they arepremoistened with 200 Kg of a percent watery metha'v nol solution, andpercolated, after standing, for 24 hours with the same solvent at roomter'nperaturehav-v ing a runoff speed of about 4 Kg per hour. A total of300 Kg of extract were obtained. The extract was brought to a pH of 9with a suspension of 250 g of calcium hydroxide in a little water,theprecipitate was filtered off, and the liquid was evaporated, in avacuum, to a thick syrup. About 75 Kg of syrup containing 80 percent ofsolid substance was obtained. Accompanied by forceful stirring, 30 Kg ofmethanol wereintroduced into a thin jet and the almost clear solution,was cooled down to 15C. Accompanied by furtherforceful stirring, acooled off mixture consisting of 3 liters of concentrated hydrochloricacid and 8 Kg of methanol was added. As a result, Sinistrin wasprecipitated as a viscous, nacreously-brilliant, yellowish-white mass.The solution, which was clear yellow after standing briefly, was pouredoff as completely as possible. It contained .salts, fructose andlow-molecular oligosaccharides.

Methanol can then be recovered from it by distillation andrectification. I

' The Sinistrin precipitate, about 40 Kg, was forcefully kneaded threetimes with 40 Kg of methanol, each 4 time, in a kneading machine. Thesupernatant methanol was poured off in each case and was combined withthe one obtained during the firstprecipitation, whereby the entire masswas converted into a fine-grained cyrstallin'e suspension. This wascontrifuged on a centrifuge and the substance isolated (about 35 Kg).wasdissolved in 70 Kg of water. The solution was then stirred with 3 Kg ofactive carbon, filtered, and it was then moved at about 15 liters perhour over a mixing bed consisting of 5 liters of a slightly acid cationexchanger in the PH form and 10 liters of a strongly basic anionexchanger in the 01-1 form. The Rein-Sinistrin solution thus obtainedwas concentrated into'a thick syrup (in tary power: i I

[a] =39.2 (c 10, H O). Yield: 31.5 Kg.

' EXAMPLE 2 Production of Polyfructosan From Garlic (Allium I Sativum)Stalks, roots, and external dry skins were stripped coarsely from 100 Kgof garlic and the rest was run through a meat grinder. The pap wasimmediatelyforcefully mixed with 30 Kg of isopropanol and waspercolated, after standing about 12 hours, with a 30% watery isopropanolsolution, at a runoff speed of about 2.5 Kg per hour. A total of 200 Kgof extract was obtained. Further processing was accomplished exactly inthe manner of Example 1. Yield: 10.5 Kg. Optical rotary power:

[a] 40.5 (c= 10, B 0). The other properties exhibited were as describedin Example 1.

Although the present invention has been adequately described in theforegoing specification and'examples included therein, it is readilyapparent that various changes and modifications can be made withoutdeparting from the spirit and scope of the invention.

c. filtering and concentrating said extract into a thick syrup underreduced pressure,

d. adding to said extract, in an amount equal to about half of saidextracts weight, a low alcohol,

e. acidifying said extract with a strong acid, to obtain .a pH ofbetween 1 and 3,

f. subsequently precipitatingthe polyfructosan with a member selectedfrom the goup consisting of a low alcohol and acetone, in an amount of 2to 8 times the weight of said extract, and

g. converting the precipitate thus formed into a powdery crystallinestate by repeatedly kneading the same with a water-free precipitationagent.

2. The process of claim ll, further comprising the step of desalting thepolyfructosan thus obtained by means of ion exchange.

3. The process of claim 2, further comprising the step of filtering thethus desalted polyfructosan with active carbon.

4. The process of claim 3, further comprising the step of removing theorganic solvent residues remaining through distillation, or drying saidpolyfructosan after concentration, into a syrup consistency in a vacuumat a maximum temperature of 80C.

5. The process of claim 1, wherein said low alcohol vacuum at a maximumtemperature of C.

1. A METHOD FOR OBTAINING THE HIGLY WATER-SOLUBLE POLYFRUCTOSANS FROMTHE STORAGE ORGANS OF MONOCOTYLEDON PLANTS, WHICH COMPRISES, A.EXTRACTING SAID POLYFRUCTOSAN WITH A MEMBER SELECTED FROM THE GROUPCONSISTING OF A LOW ALCOHOL AND ACETONE HAVING A WATER CONTENT OF FROM80-40 PERCENT, B. BRINGING SAID EXTRACT TO A PH OF ABOUT 9 WITH CALCIUMHYDROXIDE, C. FILTERING AND CONCENTRATING SAID EXTRACT INTO A THICKSYRUP UNDER REDUCED PRESSURE, D. ADDING TO SAID EXTRACT, IN AN AMOUNTEQUAL TO ABOUT HALF OF SAID EXTRACT''S WEIGHT, A LOW ALCOHOL, E.ACIDIFYING SAID EXTRACT WITH A STRONG ACID, TO OBTAIN A PH OF BETWEEN 1TO 3, F. SUBSEQUENTLY PRECIPITATING THE POLYFRUCTOSAN WITH A MEMBERSELECTED FROM THE GROUP CONSISTING OF A LOW ALCOHOL AND ACETONE, IN ANAMOUNT OF 2 TO 8 TIMES THE WEIGHT OF SAID EXTRACT, AND G. CONVERTING THEPRECIPITATE THUS FORMED INTO A POWDERY CRYSTALLINE STATE BY REPEATEDLYKNEADING THE SAME WITH A WATER-FREE PRECIPITATION AGENT.
 2. The processof claim 1, further comprising the step of desalting the polyfructosanthus obtained by means of ion exchange.
 3. The process of claim 2,further comprising the step of filtering the thus desalted polyfructosanwith active carbon.
 4. The process of claim 3, further comprising thestep of removing the organic solvent residues remaining throughdistillation, or drying said polyfructosan after concentration, into asyrup consistency in a vacuum at a maximum temperature of 80*C.
 5. Theprocess of claim 1, wherein said low alcohol is a member selected fromthe group consisting of methanol, ethanol and isopropanol.
 6. Theprocess of claim 1, wherein said strong acid is HCl.
 7. A method forpurifying the polyfructosan obtained in claim 1, which comprises: a.desalting the polyfructosan thus obtained by means of ion exchange, b.filtering the thus obtained desalted polyfructosan with active carbon,and c. removing the organic solvent resiDues remaining throughdistillation, or drying said polyfructosan after concentration, into asyrup consistency in a vacuum at a maximum temperature of 80*C.